Voltage-dependent sodium channels and calcium-activated potassium channels from mammalian brain will be studied by a combination of biochemical, biophysical and electrophysiological techniques. The general goal is to relate the molecular structures of these channels to their function in a defined membrane environment (planar lipid bilayer) and in situ (in intact mammalian neurons). Emphasis will be placed on possible modes of modulation by physiological intracellular messengers such as calcium and cyclic nucleotides and by pharmacological agents. Purified voltage-dependent sodium channels will be reconstituted in planar lipid bilayers and in phospholipid vesicles. The voltage dependence of block by saxitoxin (STX) and tetrodotoxin will be investigated in reconstituted vesicles using binding of 3H-STX. The STX binding site will be modified with carboxyl-specific reagents that allow control of the size and charge of the added group. The functional consequences of modification will be monitored in planar bilayers. Radioactive and fluorescent probes will be incorporated into the STX binding site, the former serving as a marker for the peptide containing the binding site carboxyl residue, the latter providing an optical probe to report suspected conformational changes in the structure of the binding site. Modulation of calcium-activated potassium channels by phosphorylation via protein kinases and block by phenothiazine and butyrophenone neuroleptic drugs will be studied in planar bilayers. Specific binding by phenothiazines and charybdotoxin will be used to develop a strategy for channel purification. The role of these calcium-activated potassium channels in intact cells and neural networks will be studied in brain slices and in isolated pyramidal cells by patch clamp and intracellular recording techniques.